cytogenetic biomarkers of ionising radiation exposure: a ... · 5 past exposure to densely ionising...
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Cytogenetic biomarkers of ionising radiation exposure: a multiparametric approach
M. Prakash HANDE, Ph.D., M.P.H.
Genome Stability Laboratory Department of Physiology
Yong Loo Lin School of Medicine National University of Singapore
Singapore
Genome Stability Laboratory @ Department of Physiology
• Telomeres and telomerase • DNA damage response and repair • Experimental Cancer Therapeutics • Radiation Biology/Biodosimetry • Toxicogenomics/Environmental
Toxicology
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Frequencies of dicentrics and genomic translocations in mouse splenocytes following whole body, 2 Gy X-irradiation
Genomic Translocations Dicentrics
Days after exposure
Freq
uenc
y pe
r cel
l
Hande et al. (1996a) Int. J. Radiat. Biol. 69:437-446
D(t)=0.30 e-0.05t
T(t)=0.35 e-0.006t
0.50
0.00
0.10
0.20
0.30
0.40
-20 0 80 60 20 40 120 100
d t
f
Days after exposure
Freq
uenc
y pe
r 100
cel
ls
Translocations in mouse splenocytes after in vivo X-irradiation analysed by FISH
0 10
20
30
40
50
60
0 50 100 150 200 250
Hande and Natarajan (1998) Int. J. Radiat. Biol. 74, 441-448
Hande and Natarajan (2009) In: Radiation Risk Health Sciences, Springer Japan. Pp 143-149.
0.5 Gy 1.0 Gy 2.0 Gy 3.0 Gy
0
10
20
30
40
50
60
0 50 100 150 200 250
Reciprocal Non-reciprocal
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Frequencies of micronuclei and fragments in mouse splenocytes following 2 Gy X-irradiation
Freq
uenc
y pe
r 100
cel
ls
Days after exposure
Hande et al. (1996) Int. J. Radiat.Biol. 70, 375-383
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Detection of intra-chromosomal exchanges in human lymphocytes
Boei et al. (1999) Mutat Res 404: 45-53
April 2001 – Ozeorsk, Russia MAYAK PRODUCTION ASSOCIATION
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Past exposure to densely ionising radiation leaves a unique permanent signature in the genome
Intrachromosomal aberrations: pericentric, paracentric inversions and intra-arm inversions measured in chromosome 5; Interchromosomal aberrations: Apparently simple translocations between heterologous pair of chromosome
Hande et al (2003) Am J Hum Genet 72:1162-1170
Simple versus Complex Chromosomal exchanges in Mayak workers
0 2 4 6 8 2 4 6 8
Controls
Reactor Workers Zero Pu, High γ
Moderately exposed Pu workers
Highly exposed Pu workers
Aberration Frequency (%)
Simple Complex
82/1414
10/444
67/1424
4/582
41/1414
1/444
3/1424
0/582
88/1414*
3/444*
7/1424*
0/582*
*total aberrations from complex aberrations Hande et al. 2005 Genes Chromosomes Cancer. 2005 Sep;44(1):1-9
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Complex Chromosome Aberrations in Mayak Workers
Hande et al. 2005 Genes Chromosomes Cancer. 2005 Sep;44(1):1-9
Approach
• Individual end points were studied independently by different laboratories
• Few or no attempts were made to study different biomarkers on a single sample
• Need for a multi-parametric approach on a single sample following irradiation
Initiated the work in 2006
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Ionising Radiation
Gamma radiation
Whole blood (A)
Peripheral blood derived
lymphocytes (B)
Heavy Ion Radiation**
Carbon (whole blood)
(C)
Iron (whole blood)
(D)
(n=10) γH2AX cronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression Cytokine profile
(n=10) γH2AX Micronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression miRNA expression Proteomic profile
(n=3) γH2AX Micronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression
(n=3) Micronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression
Doses : 0.1 Gy, 0.25 Gy, 0.5 Gy, 1 Gy (1.06 Gy/min, 137Cs source)
Time points post irradiation : 2 h and 24 h
**National Institute of Radiological Sciences, Chiba, Japan (R Okayasu)
Micronuclei detection in mouse/human cells
Hande et al. (1996) Int. J. Radiat.Biol. 70, 375-383 Poonepalli et al. (2005) Cancer Research 65: 10977-109883
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Single and Two colour FISH
Interphase nuclei: Single colour and two colour FISH
Minor satellite Lambda DNA and c-myc gene
Cop
yrig
ht ©
199
4 M
. Pra
kash
Han
de
Cop
yrig
ht ©
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4 M
. Pra
kash
Han
de
Frequency of centromere-positive micronucleus in mouse splenocytes
Hande et al. (1996) Int. J. Radiat.Biol. 70, 375-383
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PNA FISH in the detection of telomeres
Blasco, Lee, Hande et al. 1997, Cell 91:25-34
Use of PNA FISH with telomere probe in mouse studies
Hande et al. 1999 J. Cell Biol.144: 589-601
Di fagagna, Hande et al. 1999 Nature Genetics
Di Fagagna, Hande et al., 2001 Current Biology
RLC
Dicentric
RLC
translocation
Translocation
Dicentric
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Use of centromere and telomere PNA probes to identify chromosome aberrations in human cells
Low et al. 2008 Int J Biochem Cell Biol 40:2583 – 2595
Gopalakrishnan et al. 2010 Genome Integr 1:16
Vinoth et al. 20008 Stem Cells Dev 17:599 – 607
Banerjee et al 2008 Breast Cancer Res Treat 107: 25 -31
Boei, Vermeulen, Natarajan 2000 Int J Radiat Biol 76:163-167
Differential gene
expression profile
following irradiation: two
way hierarchical
clustering
0 0.1 0.25 0.5 0 0.1 0.25 0.5
0.25 Gy
24 h
Radiation Dose (Gy)
2 h
3 ANOVA (p<0.05)
ROW : GENE COLUMN : SAMPLE
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Gene expression profile following gamma irradiation
PBMCs Principle component analysis
Whole Blood
High-throughput expression profiles of
transcriptome, miRNA, etc
Time/dose of known radiation exposure
Dose-response following exposure to
human peripheral Blood (ex vivo)
Dose-response clinical endpoints of all parameters
Biological data from public
domains and other literatures
Low, medium & high-throughput profiles of
radiation exposure
Risk estimation, elucidation of underlying
mechanism
Transcriptomic Knowledgebase
REFIRE Knowledgebase
Transcriptomics miRNA Cytogenetics
Risk Estimation Following (low-dose) Ionising Radiation Exposure (REFIRE)
Jayapal, Zeegers, Sethu, Banerjee, Hande 2011 Defence Science Journal, Vol. 61, 133-137
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REFIRE Knowledgebase - Our Tool Box for Radiation Biology
Dose, Time
Networks
Bioinformatics analysis
MN, CA DNA damage
Today
Optimised Dose, time catalogue
Complex pathways
Advance complex analysis
databases
Dose,/Time based catalogue
of MN and CA
Tomorrow
Biomarkers
Catalogue of Time / dose
specific markers
From a basic toolbox today to…
Integrated design, measurements & tools to enable predictable radiation exposure
Pilot population
Broad population spectrum
Summary
• Donor specific effects and individual variation should be taken in to consideration for biomarker identification
• Gene expression – changes may depend on time after exposure than on dose
• More than one biological response marker may be necessary in biodosimetry
• Influence of life-style factors
• Validation of these biomarkers will be done in a larger population and radiotherapy patients
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Study design for CRP
• To generate calibration curves for gamma radiation in human blood lymphocytes
• Human PBL • 3 apparently healthy donors • 0, 0.25, 0.5, 1, 2, 3 and 4 Gy; 137Cs • Dicentric and Micronucleus assay
Acknowledgements
Defence Innovative Research Programme, Defence Science and Technology Agency, Singapore; IAEA CRP E3. 50.08
• Swaminathan Sethu • Grace Low • Aik Kia Khaw • Birendranath Banerjee • Dimphy Zeegers • Manikandan Jayapal
• Susan Loong Li Er, National Cancer Centre, SGH, Singapore
• Rajamanickam Baskar (Radiation Oncology, National Cancer Centre, Singapore)
Ryuichi Okayasu and Takamitsu Kato, NIRS, Chiba